Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop

Abstract

The Abl tyrosine kinase inhibitor STI-571 is effective therapy for stable phase chronic myeloid leukemia (CML) patients, but the majority of CML blast-crisis patients that respond to STI-571 relapse because of reactivation of Bcr-Abl signaling. Mutations of Thr-315 in the Abl kinase domain to Ile (T315I) were previously described in STI-571-resistant patients and likely cause resistance from steric interference with drug binding. Here we identify mutations of Tyr-253 in the nucleotide-binding (P) loop of the Abl kinase domain to Phe or His in patients with advanced CML and acquired STI-571 resistance. Bcr-Abl Y253F demonstrated intermediate resistance to STI-571 in vitro and in vivo when compared with Bcr-Abl T315I. The response of Abl proteins to STI-571 was influenced by the regulatory state of the kinase and by tyrosine phosphorylation. The sensitivity of purified c-Abl to STI-571 was increased by a dysregulating mutation (P112L) in the Src homology 3 domain of Abl but decreased by phosphorylation at the regulatory Tyr-393. In contrast, the Y253F mutation dysregulated c-Abl and conferred intrinsic but not absolute resistance to STI-571 that was independent of Tyr-393 phosphorylation. The Abl P-loop is a second target for mutations that confer resistance to STI-571 in advanced CML, and the Y253F mutation may impair the induced-fit interaction of STI-571 with the Abl catalytic domain rather than sterically blocking binding of the drug. Because clinical resistance induced by the Y253F mutation might be overcome by dose escalation of STI-571, molecular genotyping of STI-571-resistant patients may provide information useful for rational therapeutic management.

Fig 1.

The Bcr-Abl Y253F mutant has intermediate in vivo resistance to STI-571 relative to the T315I mutant. (A) 293 cells transfected with Bcr-Abl WT or the Y253F or T315I mutants were treated for 2 h with increasing doses of STI-571, and cell lysates were analyzed by immunoblotting with anti-Abl (Top) and antiphosphotyrosine (Bottom) antibodies. (B) Ba/F3 cells expressing Bcr-Abl WT (blue symbols), Bcr-Abl Y253F (red symbols), or Bcr-Abl T315I (orange symbols) were incubated for 24 h in the indicated concentrations of STI-571 in the absence of IL-3, and viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye-reduction assay (11). Results are plotted as percentage of cells incubated without STI-571. As a control for toxicity, Bcr-Abl WT-expressing cells grown in the presence of IL-3 (black symbols) are included. (C) Anti-Abl immunoprecipitates (IP) from the cell lines in B were subjected to in vitro kinase assay with a GST-Crk substrate in the presence of the indicated concentration of STI-571 as described (12); incorporation of ³²P into the substrate was determined by autoradiography (Top and Middle), whereas the GST-Crk protein levels were demonstrated by Coomassie blue (CB) staining (Bottom) in a representative of three independent experiments. (D) Data from C were quantitated by PhosphorImager and densitometric analysis, and sigmoidal plots were generated for the calculation of IC₅₀ values as described in Materials and Methods. Error bars indicate standard deviation.

Fig 2.

The Y253F mutation dysregulates c-Abl and induces resistance to STI-571 in vitro and in vivo. (A) Anti-Abl (Upper) and antiphosphotyrosine (Lower) immunoblots of lysates from parental Ba/F3 cells (par) and populations stably overexpressing c-Abl WT, P112L, and Y253F proteins. (B) Anti-Abl immunoprecipitates (IP) from the lysates in A were subjected to in vitro kinase assay as described for Fig. 1C, with incorporation of ³²P into the Crk substrate determined by autoradiography (Upper) and GST-Crk protein levels demonstrated by Coomassie blue (CB) staining (Lower) in a representative of two independent experiments. (C) Data from B were quantitated by PhosphorImager and densitometric analysis, and sigmoidal plots were generated for the calculation of IC₅₀ values. (D) Ba/F3 cells expressing c-Abl P112L (green symbols) or c-Abl Y253F (red symbols) were assessed for their response to STI-571 as described for Fig. 1B. (E) Photomicrographs of transformed NIH 3T3 cells stably expressing c-Abl P112L (Upper) and c-Abl Y253F (Lower), incubated 24 h in the indicated concentration of STI-571. (Magnification, ×250.) (F) Antiphosphotyrosine (Upper) and anti-Abl (Lower) immunoblots of lysates from the cells in E. Molecular mass markers are shown on the left.

Fig 3.

The Y253F mutation does not dysregulate the catalytic activity of c-Abl in vitro. (A) Purified c-Abl WT, P112L, and Y253F proteins have undetectable phosphotyrosine but rapidly autophosphorylate upon incubation in the presence of ATP and magnesium for the indicated reaction (rxn) time (9). (Upper) Antiphosphotyrosine blot. (Lower) Anti-Abl blot. (B) Michaelis–Menten (Left) and Lineweaver–Burke (Right) plots of the catalytic activity of c-Abl WT (blue), P112L (green), and Y253F (red) in solution in the peptide kinase assay.

Fig 4.

The Abl Y253F mutant has intrinsic resistance to STI-571 that is independent of phosphorylation of Tyr-393. (A) Sensitivity of purified unphosphorylated c-Abl WT kinase to STI-571 at different concentrations of ATP (red, 100 μM; green, 200 μM; blue, 500 μM). (Inset) Data from these ATP concentrations and a 50 μM ATP series (not shown) were plotted in a double-reciprocal format [x axis: 1/(ATP)(μM⁻¹); y axis: 1/v(min⋅pmol⁻¹) for STI-571 concentrations of 0.1, 0.3, and 1.0 μM (as indicated)]. (B) Sensitivity of c-Abl to STI-571 at different concentrations of magnesium chloride (red, 1 μM; green, 5 μM; blue, 10 μM). (C) Sensitivity of purified unphosphorylated c-Abl WT (blue), P112L (green), and Y253F (red) to STI-571. (D–F) Comparison of the STI-571 sensitivity of unphosphorylated (black symbols) and tyrosine-phosphorylated (red symbols) c-Abl WT (D), P112L (E), and Y253F (F). The STI-571 sensitivity of an unphosphorylated Y253F/Y393F double mutant is shown in green symbols in F. The Y393F mutation alone did not alter the STI-571 sensitivity of c-Abl significantly (IC₅₀ = 1.40, blue symbols in F), and the resistance of c-Abl Y393F to STI-571 was not increased by phosphorylation (data not shown). A modest stimulatory effect of STI-571 on peptide kinase activity of phosphorylated c-Abl and c-Abl Y253F was reproducibly observed at lower drug concentrations.